There has been a dramatic increase in allogeneic hematopoietic transplant activity at the Clinical Center during the past 2 years, due to markedly increased numbers of peripheral blood stem cell (PBSC) transplants performed by NHLBI and NCI investigators. Nonmyeloablative transplant protocols piloted by NHLBI have targeted large numbers of CD34-positive stem cells in the donor graft, in order to (1) facilitate rapid and durable engraftment, (2) promote graft-versus-tumor activity, (3) hasten immunologic reconstitution, and (4) permit complex graft engineering. Meeting target PBSC CD34 cell doses requires large volume leukapheresis (LVL) in healthy donors. To optimize use of limited space and staff resources, and minimize donor discomfort and inconvenience, we studied the kinetics of CD34 mobilization, the apheresis product yield, and degree of resource utilization in very large volume vs standard volume leukapheresis. Allogeneic donors were randomly assigned to a single 25-liter PBSC collection or two consecutive daily 15L PBSC procedures. Donors received filgrastim 10 (g/kg/d SC for 5 (25 L x 1) or 6 days (15 L x 2). LVL was performed using a Baxter CS-3000 Plus with blood flow rate of 80-85 ml/min, ACDA ratio of 13:1, and prophylactic intravenous calcium chloride infusion. Blood CD34 counts were measured prior to, after each 5 L processed, and 1 hour after LVL. Seven donors were studied in each group. Donor gender, age, weight, and blood volume were similar in the two groups. However, day 5 preapheresis CD34 counts were higher in the group undergoing 15L LVL x 2 (78 vs 62 cells/(l, p less than 0.05). Despite the higher initial blood CD34 count, the overall CD34 cell yield in the single 25 L procedure that same as that in the two consecutive 15 L procedures (750 vs 820 million CD34 cells, p greater than 0.05). The minimum target dose of 3 x10 to the sixth power CD34 kg recipient weight was met in 7/7 25L x 1 and 6/7 15L x 2 procedures. Circulating CD34 counts decreased by 25 to 30 percent at 15L processed, but increased above pre-apheresis values one hour following LVL in all procedures. Day 5 lymphocyte counts prior to apheresis, and total lymphocyte yield, were the same in both groups. No donor in either group experienced citrate-related symptoms or other adverse effects of apheresis. These studies demonstrate that a single 25-L large volume leukapheresis for allogeneic PBSC collection results in similar CD34 product yields as two consecutive daily 15-liter procedures, but is more convenient for the donor, requires one rather than two hospital visits, less venipunctures, and one less dose of filgrastim. In addition, the single 25-L procedure significantly decreased apheresis staff time and space utilization and halved the costs of apheresis supplies and laboratory testing. These results have had substantial impact in changing DTM policies for the performance of LVL for allogeneic PBSC collection in both related sibling and matched unrelated donor settings.